This invention relates to the fractionation of heterogeneous populations of cells or the like to isolate a relatively homogeneous sub-population of a specific cell type. More specifically, the improvement of this invention relates to magnetic sorting of cells, bacteria, or viruses.
A general procedure for magnetic sorting of cells, bacteria, and viruses is disclosed in U.S. Pat. No. 3,970,518, issued July 20, 1976. In that procedure, uncoated particles of a magnetic material, such as iron oxide, are contacted with a high concentration liquid dispersion of the selective antibody, and after sufficient antibody has adhered to the magnetic particles, the coated particles are contacted with the mixed population to be fractionated, the select cell or the like binding to the magnetic particles, and the bound cells are then separated magnetically from the remainder of the population. As a further step, the select cells may be separated from the magnetic material, by the use of a cleaving agent solution and magnetic removal of the magnetic particles.
While there are literature reports describing the use of magnetic microspheres in cell sorting, there is no literature verification that uncoated magnetic particles can be made to effectively bind with antibodies. In the published procedures, the particles of magnetic material are contained in microspheres formed from polymers, which can be chemically coupled to antibodies. See, for example: Molday et al, Nature, 268, 437 (1977); Kronick et al, Science, 200, 1074 (1978); and Antoine et al, Immunochemistry, 15, 443 (1978). These references describe magnetically-responsive microspheres formed from acrylate polymers, such as hydroxyethyl methacrylate, or polyacrylamide-agarose microspheres. Such microspheres can be chemically coupled to antibodies with glutaraldehyde or other di-aldehyde. As described by the cited Molday (1977) and Kornick references, one procedure involves the chemical attachment of diaminoheptane spacer groups to the microspheres, which are then chemically linked to the antibodies by glutaraldehyde reaction. Although effective bonding of the antibodies can be obtained, such procedures are difficult since aggregation of microspheres can readily occur and the preparative procedure is time consuming. For example, the reaction to attach spacer groups may require from five to twelve hours of chemical reaction time, and subsequent dialysis to remove the excess reagent. The coupling of the antibodies may then require another twelve to twenty-four hours followed by dialysis to remove excess coupling agent. Further, such antibody reagents may not be used efficiently, since an excess of the antibodies will usually need to be present during the chemical coupling.
Another disadvantage of magnetic particle or microsphere separation methods as described in the art is that the antibodies are attached to the microspheres in a random manner. Antigen-binding occurs through the Fab regions of the antibodies which are in the outer portions of the arms. With random attachment of the antibodies, one or both of the Fab arms may be unavailable for antigen-binding. thus, an excess of antibody must be used to assure that the coated microspheres effectively bind to the antigens associated with the cells or other bodies being sorted.